JP2014517023A - Peptide extraction method and its use in liquid phase peptide synthesis - Google Patents

Abstract

The present invention is a method for extracting a peptide from a reaction mixture obtained by a peptide coupling reaction,
The reaction mixture comprises a peptide and a polar aprotic solvent selected from the group consisting of N, N-dimethylformamide, N, N-dimethylacetamide and N-methyl-2-pyrrolidone; ,
a) adding to the reaction mixture component a1) which is toluene and component a2) which is water to obtain a two-phase system consisting of an organic layer and an aqueous layer, and b) an organic layer containing a peptide The present invention relates to a method comprising a step including separating an aqueous layer.
In a particularly preferred embodiment of the present invention, the extraction method of the present invention uses a combination of an organic solvent 1 selected from the group consisting of n-heptane, 2-methyltetrahydrofuran, ethyl acetate, isopropyl acetate, acetonitrile and tetrahydrofuran and toluene. use. This extraction step is preferably used in a peptide preparation method in a liquid phase.

Description

  The present invention relates to a method for extracting a peptide from a reaction mixture obtained by a peptide coupling reaction. The method of the present invention is preferably used in a liquid phase peptide synthesis (LPPS) method. The method of the present invention for extracting a peptide from a reaction mixture can also be used in other peptide synthesis methods. For example, after cleaving a synthetic peptide prepared by the solid phase peptide synthesis (SPPS) method, Can be used when releasing. The method of the present invention can also be used in a solid-liquid phase hybrid peptide synthesis method. Furthermore, the peptide extraction method of the present invention can be used to isolate peptides from natural sources such as yeast and bacteria, specifically to isolate peptides expressed by recombinant techniques. Can be used.

  In this specification, the nomenclature of amino acids and peptides is “Nomenclature and symbolism for amino acids and peptides”, Pure & Appl. Chem. 1984, Vol. 56, No. 5, pp. 595-624 unless otherwise stated. Shall be followed.

The following abbreviations have the following meanings unless otherwise indicated.
ACN Acetonitrile
Boc tert-butoxycarbonyl
Bsmoc 1,1-Dioxobenzo [b] thiophen-2-ylmethyloxycarbonyl
Bzl benzyl
Cbz benzyloxycarbonyl
DCC N, N'-dicyclohexylcarbodiimide
DCM dichloromethane
DEA diethylamine
DIPE Diisopropyl ether
DIPEA N, N-Diisopropylethylamine
DMA N, N-dimethylacetamide
DMF N, N-dimethylformamide
EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
eq equivalent
EtOAc ethyl acetate
Fmoc Fluorenyl-9-methoxycarbonyl
h hours
HOBt 1-hydroxybenzotriazole
HOBt ・ H ₂ O 1-Hydroxybenzotriazole monohydrate
HPLS HPLC
LPPS liquid phase peptide synthesis
MeTHF 2-methyltetrahydrofuran
min minutes
MS mass spectrometry
NMP N-methyl-2-pyrrolidone
OMe Methoxy
OtBu tert-butoxy
PG protecting group
PyBOP Benzotriazol-1-yloxy-tris (pyrrolidino) -phosphonium hexafluorophosphate
SPPS solid phase peptide synthesis
TAEA tris (2-aminoethyl) amine
TBTU O- (Benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate
tBu tert-butyl
TEA Triethylamine
TFA trifluoroacetic acid
THF tetrahydrofuran
TLC thin layer chromatography
TOTU O- [cyano (ethoxycarbonyl) methyleneamino] -1,1,3,3-tetramethyluronium tetrafluoroborate
Trt Trityl
UV UV

  The peptide extraction method is usually used in various peptide synthesis methods such as liquid phase peptide synthesis (LPPS), solid phase peptide synthesis (SPPS), and solid phase-liquid phase hybrid peptide synthesis.

  LPPS is particularly often used for industrial mass production of peptides. In LPPS, usually two partially protected amino acids or peptides, ie, an amino acid or peptide having an unprotected C-terminal carboxylic acid group and an amino acid or peptide having an unprotected N-terminal amino group, To couple. After completion of the coupling step, the N-terminal amino group or C-terminal carboxylic acid group is deprotected by specifically cleaving the protecting group (PG) of the N-terminal amino group or C-terminal carboxylic acid group of the obtained peptide. And a subsequent coupling step can be performed. LPPS is usually completed by performing a comprehensive deprotection step that removes all remaining protecting groups (PG).

  It is often difficult to handle peptides, particularly peptides with unprotected C-terminal carboxylic acid groups and / or unprotected N-terminal amino groups in LPPS. This is because such peptides are difficult to dissolve in common organic solvents. In general, the solubility of peptides in common organic solvents decreases with increasing peptide chain length.

  Dichloromethane (DCM) is commonly used in LPPS as a suitable reaction solvent. Since DCM has excellent solvent properties, a low boiling point, and low miscibility with water, it is possible to perform extraction with an aqueous solution as a post-treatment of the reaction mixture by using DCM. However, if DCM is used on an industrial scale, the environment may be adversely affected. Also, because DCM has a high density, it takes time and money to extract a DCM layer using an aqueous solution. The use of DCM is usually limited.

  Furthermore, recently developed highly efficient coupling reagents such as benzotriazol-1-yloxy-tris (pyrrolidino) -phosphonium hexafluorophosphate (PyBOP) and O- (benzotriazol-1-yl) -1,1 3,3-Tetramethyluronium tetrafluoroborate (TBTU) has low solubility in DCM. These coupling reagents are particularly advantageous when coupling two large peptide fragments, and the use of other coupling reagents to couple such large peptide fragments is known to reduce yield. Yes.

  In addition, many of the peptides are difficult to dissolve in DCM under neutral and basic conditions, while N, N-dimethylformamide (DMF), N, N-dimethylacetamide (DMA), N-methyl-2-pyrrolidone, for example It dissolves well in polar aprotic solvents such as (NMP). Therefore, these polar aprotic solvents are conventionally used alone or mixed with a low polarity solvent such as tetrahydrofuran (THF) as a reaction solvent for LPPS.

  On the other hand, the use of polar aprotic solvents for LPPS has many drawbacks. Since the polar aprotic solvent has a high boiling point, it is difficult to evaporate and concentrate the reaction mixture. Furthermore, since polar aprotic solvents are miscible with water, it is not possible to perform direct extraction with an aqueous solution as a post-treatment of the reaction mixture.

  When LPPS is performed on an industrial scale, the intermediate peptide is usually precipitated directly from the reaction mixture after each coupling step. This allows separation from unreacted starting materials, by-products, excess coupling reagents and impurities such as bases. After completion of the peptide coupling reaction, the peptide is usually precipitated by adding the reaction mixture to a poor solvent (such as diethyl ether or water). However, as a known problem, it is known that a gel is formed when the reaction mixture is added to a poor solvent.

  Furthermore, since polar aprotic solvents generally inhibit the peptide precipitation process, the peptide precipitates as a sticky rubbery solid, making filtration and drying difficult. In some cases, the precipitated peptide cannot be filtered, and further, the precipitated peptide cannot be transferred onto the filter. In particular, it is often difficult to precipitate peptides on an industrial scale, requiring a lot of time, and the lead time depends on the filtration time. This problem can be partially solved by increasing the volume ratio of antisolvent to polar aprotic solvent in the precipitation step. In practice, a large amount of a suitable antisolvent is required to obtain a filterable form of the peptide precipitate.

  Furthermore, the polar aprotic solvent remaining in the peptide precipitate is known to inhibit the subsequent deprotection step using trifluoroacetic acid (TFA). Therefore, before cleaving the acid cleavable protecting group (PG), such as tert-butoxycarbonyl (Boc), trityl (Trt), tert-butyl (tBu), tert-butoxy (OtBu), etc. A further step is required to wash the peptide precipitate with a solvent that is more volatile than the protic solvent to remove the remaining polar aprotic solvent.

Related technology
WO2005 / 081711 relates to drug-linker-ligand conjugates, drug-linker compounds, and methods of their use for treating cancer, autoimmune diseases or infectious diseases. This patent document discloses a method for preparing a peptide-based drug, a method for extracting a peptide, and the like using ethyl acetate, dichloromethane and a tBuOH / CHCl ₃ mixture.

  US 5,869,454 relates to arginine keto-amide enzyme inhibitors. In this patent document, synthesis of such an inhibitor and extraction with ethyl acetate are disclosed.

  US2005 / 0165215 relates to a peptide synthesis method and a peptide isolation method in the synthesis method. This patent document further relates to improvements for large-scale synthesis of peptides. In this patent document, halogenated organic solvents such as dichloropropane, dichloroethane, dichloromethane, chloroform, chlorofluorocarbon, chlorofluorohydrocarbon, and mixtures thereof are proposed as suitable solvents for peptide extraction. A preferred solvent is dichloromethane.

  CH Schneider et al. (Int. J. Peptide Protein Res. 1980, 15, pp. 411-419) report a method for peptide synthesis in solution in which intermediate purification is performed by liquid-liquid extraction (two-phase method). . Dichloromethane is used as a peptide extraction solvent.

  J. W. van Nispen (Pure and Appl. Chem. 1987, Vol. 59, No. 3, pp. 331-344) outlines the synthesis and analysis of (poly) peptides. This document teaches that it is possible to combine various solvents with various properties in order to find optimal separation conditions for peptide components. A so-called Craig device is generally used to achieve this purpose, in which the lower phase is fixed and the upper phase is moved to distribute in multiple stages.

  US2010 / 0184952 discloses a method for degrading a dibenzofulvene and / or dibenzofulveneamine adduct from a reaction mixture obtained by deprotecting an amino acid compound protected with an Fmoc group with an amine, The mixture is stirred and separated in a hydrocarbon solvent having 5 or more carbon atoms and a polar organic solvent immiscible with the hydrocarbon solvent (excluding an amide organic solvent), and dibenzofulvene and / or dibenzofulveneamine adduct A method is disclosed that includes the step of removing the hydrocarbon solvent layer in which the is dissolved. In this method, the amino acid ester or peptide is transferred into a polar organic solvent. Examples of the polar organic solvent include acetonitrile, methanol, acetone and the like, or a mixed solvent thereof, and acetonitrile or methanol is preferable.

  L. A. Carpino et al. (Organic Process Research & Development 2003, 7, pp. 28-37) report a rapid and continuous liquid phase peptide synthesis. In the method of this document, the Fmoc protecting group and the Bsmoc protecting group of a peptide fragment are deprotected in the presence of tris (2-aminoethyl) amine, and this method is fast and continuous in grams of a short peptide. It has been identified that it can be used for liquid phase synthesis and the synthesis of relatively long (22mer) fragments (hPTH 13-34). In the latter synthesis, the purity of the crude product has been reported to be significantly higher than the sample obtained by the solid phase method. This method using Bsmoc has been optimized by incorporating a new technique for filtering partially deprotected peptides that are extended during each coupling-deprotection cycle through a silica gel short column. .

  However, the method reported by L. A. Carpino et al. Has some limitations. That is, since this method uses DCM as a reaction solvent, it cannot be used for the preparation of peptides that are sparingly soluble in DCM. Furthermore, the use of large amounts of expensive tris (2-aminoethyl) amine (TAEA) also makes it difficult to utilize this method on an industrial scale.

  Therefore, there is a strong need for time-effective and cost-effective synthetic methods for preparing peptides, especially for industrial scale production. Such a method should be able to solve the problems caused by the use of DCM in LPPS and polar aprotic solvents such as DMF, DMA, NMP.

  Surprisingly, the inventors have shown that many of the structurally diverse peptides exhibit excellent solubility in toluene, and that toluene is n-heptane, 2-methyltetrahydrofuran, ethyl acetate, isopropyl acetate, It has been found that it is preferable to use in combination with an organic solvent selected from the group consisting of acetonitrile and tetrahydrofuran (this group is referred to as “organic solvent 1”). Specifically, the solubility of peptides in toluene combined with organic solvent 1 is generally higher than that in toluene alone. Furthermore, the present inventors have found that in a two-phase system containing toluene and water combined with toluene or organic solvent 1, most of the common polar aprotic solvent is transferred to the aqueous layer.

  Therefore, a system containing toluene and water alone or in combination with organic solvent 1 is very suitable for the extraction of peptides from mixtures containing polar aprotic solvents. In one embodiment of the present invention, the organic layer obtained by partially evaporating the peptide-containing organic layer and then adding an appropriate anti-solvent (this group of solvents is referred to as “organic solvent 2”) Peptides dissolved in are precipitated. Since the polar aprotic solvent is substantially not contained when the peptide is precipitated, the obtained peptide can be easily filtered. By using the extraction method of the present invention, the time required for peptide filtration can be significantly reduced. Therefore, by using the extraction method of the present invention, the problem of LPPS caused by using a polar aprotic solvent can be solved brilliantly.

The present invention is a method for extracting a peptide from a reaction mixture obtained by a peptide coupling reaction,
The reaction mixture comprises a peptide and a polar aprotic solvent selected from the group consisting of N, N-dimethylformamide, N, N-dimethylacetamide and N-methyl-2-pyrrolidone;
The method is
a) adding to the reaction mixture component a1) which is toluene and component a2) which is water to obtain a two-phase system consisting of an organic layer and an aqueous layer, and b) an organic layer containing a peptide Including a step comprising separating the aqueous layer; and the volume composition ratio of the two-phase system obtained in step a)
The method is characterized in that polar aprotic solvent: toluene = 1: 20 to 1: 2 and polar aprotic solvent: water = 1: 20 to 1: 2.

A preferred embodiment of the present invention is a method for extracting a peptide from a reaction mixture obtained by a peptide coupling reaction,
The reaction mixture comprises a peptide and a polar aprotic solvent selected from the group consisting of N, N-dimethylformamide, N, N-dimethylacetamide and N-methyl-2-pyrrolidone;
The method is
a) Component a3 which is an organic solvent 1 selected from the group consisting of component a1) which is toluene, component a2) which is water and n-heptane, 2-methyltetrahydrofuran, ethyl acetate, isopropyl acetate, acetonitrile and tetrahydrofuran And b) separating the organic layer containing the peptide and the aqueous layer; and b) separating the organic layer and the aqueous layer comprising the peptide; The volume composition ratio of the obtained two-phase system is
Polar aprotic solvent: toluene = 1: 20-1: 2,
Polar aprotic solvent: organic solvent 1 = 1: 5-30: 1
The method is characterized in that polar aprotic solvent: water = 1: 20 to 1: 2 and toluene: organic solvent 1 = 50: 1 to 1: 1.

In a preferred embodiment, the volume composition ratio of the two-phase system obtained in step a) is
Polar aprotic solvent: toluene = 1: 6 to 1: 3
Polar aprotic solvent: organic solvent 1 = 1: 1 to 4: 1
Polar aprotic solvent: water = 1: 5 to 1: 3, and toluene: organic solvent 1 = 10: 1 to 2: 1.

  In a particularly preferred embodiment, the polar aprotic solvent is N, N-dimethylformamide or N-methyl-2-pyrrolidone.

  In yet another embodiment of the invention, the two-phase system does not contain organic solvent 1.

  In a preferred embodiment of the present invention, the peptide precipitation operation is not performed every time the peptide is extracted. That is, one or more protecting groups of the peptide are cleaved, and the resulting partially unprotected peptide is extracted, and the organic layer containing the peptide is used for the next peptide coupling reaction. Thus, the present invention provides an efficient synthetic method for performing continuous LPPS suitable for the preparation of peptides on an industrial scale.

  The continuous LPPS of the present invention is very suitable for peptide synthesis using Boc, Fmoc and Bzl as protecting groups, as described in the examples below.

It is a graph which shows the influence of residual DMF with respect to the removal rate of the Boc protective group of peptide Boc-Pro-Ile-Leu-Pro-Pro-Glu (OBzl) -Glu (OBzl) -Tyr (Bzl) -Leu-OBzl. Photo of peptide Boc-Ser (Bzl) -Phe-Pro-Ile-Leu-Pro-Pro-Glu (OBzl) -Glu (OBzl) -Tyr (Bzl) -Leu (OBzl) precipitated in the absence of DMF It is.

Extraction method The present invention is a method of extracting a peptide from a reaction mixture obtained by a peptide coupling reaction,
The reaction mixture comprises a peptide and a polar aprotic solvent; and the method comprises:
a) adding to the reaction mixture component a1) which is toluene and component a2) which is water to obtain a two-phase system consisting of an organic layer and an aqueous layer, and b) an organic layer containing a peptide The present invention relates to a method comprising a step including separating an aqueous layer.

A preferred embodiment of the present invention is a method for extracting a peptide from a reaction mixture obtained by a peptide coupling reaction,
The reaction mixture comprises a peptide and a polar aprotic solvent selected from the group consisting of DMF, DMA and NMP; and the method comprises:
a) Component a3 which is an organic solvent 1 selected from the group consisting of component a1) which is toluene, component a2) which is water and n-heptane, 2-methyltetrahydrofuran, ethyl acetate, isopropyl acetate, acetonitrile and tetrahydrofuran ) To obtain a two-phase system consisting of an organic layer and an aqueous layer, and b) a step comprising separating the organic layer containing the peptide from the aqueous layer. .

  Component a1), component a2) and component a3) may be mixed, and this mixing may be performed in any order. As long as the peptide does not precipitate in the extraction method of the present invention, these three components can be added after mixing two components or all three components in advance.

  The mixture containing a polar aprotic solvent is preferably a crude reaction mixture obtained by a peptide coupling reaction. If a compound that acts as a surfactant is contained in this mixture, phase separation in the extraction method of the present invention can be inhibited. Therefore, it is preferable that this mixture does not contain such a compound at all. In a particularly preferred embodiment, the mixture does not contain surfactants known in the prior art such as cationic surfactants and nonionic surfactants.

  Addition of component a1), component a2) and component a3) to the mixture containing the peptide and polar aprotic solvent may be performed in any order as long as the peptide does not precipitate in the extraction method of the present invention. For example, a mixture containing a peptide and a polar aprotic solvent can be mixed with toluene, water can be added thereto, and finally the organic solvent 1 can be added. Moreover, toluene and the organic solvent 1 can also be added after adding the mixture containing a peptide and a polar aprotic solvent to water.

  In a particularly preferred embodiment of the present invention, a mixture containing a peptide and a polar aprotic solvent is mixed with toluene and the organic solvent 1, which may be added first. Then add water.

  It is understood that the added water (component a2)) may contain dissolved components such as salts (eg, inorganic salts).

  The resulting two-phase system is preferably stirred vigorously. Stirring of the resulting two-phase system can be performed using a mixer commonly used in extraction operations and known in the art. For example, when performing batch extraction, a two-phase system can be stirred using a jet mixer or a stirring mixer.

  The equipment suitable for the extraction operation is selected mainly depending on the scale for performing the extraction method of the present invention and the extraction temperature. In addition, the extraction method of the present invention can be performed using a batch extraction method or a continuous extraction method. In order to achieve optimal extraction for peptides, the extraction method of the present invention can be repeated multiple times as necessary.

  After stirring, the phases are preferably separated to form two liquid layers (an organic layer and an aqueous layer). The organic layer is less dense than the aqueous layer. Phase separation may occur using a settling tube or by centrifugation. The time required for phase separation depends on the scale for carrying out the extraction method of the invention and the equipment used. The time required for phase separation is preferably less than 1 hour, more preferably less than 10 minutes, and particularly preferably less than 1 minute.

  After phase separation occurs, the peptide is mainly contained in the organic layer, which further contains toluene, and optionally further contains the organic solvent 1. The upper organic layer containing the peptide is separated from the aqueous layer. After performing the extraction method of the present invention, it is preferable that more than 90% by weight of the peptide is contained in the organic layer and less than 10% by weight is contained in the aqueous layer. Even more preferably, after performing the extraction method of the present invention, more than 98% by weight of the total peptide is contained in the organic layer and less than 2% by weight is contained in the aqueous layer. After the extraction method of the present invention is carried out, it is particularly preferred that more than 99% by weight of the peptide is contained in the organic layer and less than 1% by weight is contained in the aqueous layer.

  By the extraction method of the present invention, the peptide can be efficiently extracted from the crude reaction mixture obtained by the peptide coupling reaction. The solubility of the polar aprotic solvent in the organic layer is significantly lower than that in the aqueous layer. Thus, after extraction, the organic layer containing the peptide contains only a small amount of polar aprotic solvent.

  After carrying out the extraction method of the present invention, less than 15% by volume of the whole polar aprotic solvent is contained in the organic layer, and more than 85% by volume of the whole polar aprotic solvent is contained in the aqueous layer. It is preferred that Furthermore, after performing the extraction method of the present invention, less than 5% by volume of the whole polar aprotic solvent is contained in the organic layer, and more than 95% by volume of the whole polar aprotic solvent is contained in the aqueous layer. It is more preferable that it is contained in. After performing the extraction method of the present invention, less than 2% by volume of the total polar aprotic solvent is contained in the organic layer, and more than 98% by volume of the total polar aprotic solvent is contained in the aqueous layer. It is particularly preferred that Repeated extraction may be required to achieve this condition.

  Importantly, the extraction method according to the present invention can not only separate peptides from the majority of polar aprotic solvents, but also from salts and by-products derived from coupling reagents (urea, tetrafluoroborate, etc.). Peptides can be separated. When a hydrophobic antisolvent such as n-heptane or diethyl ether is added to the crude reaction mixture obtained by the peptide coupling reaction to precipitate the peptide directly, salts and by-products cannot usually be removed. In addition, these salts and by-products are known to reduce the performance of chromatographic columns used for peptide workup. When the prepared peptide is used as an active pharmaceutical ingredient, it is essential to further purify by column chromatography.

  Therefore, the peptide may be precipitated and then purified by column chromatography as necessary. Such a purification step is further added when the peptide is used as an active pharmaceutical ingredient. Therefore, the extraction method according to the present invention can isolate peptides with higher purity than the method of directly precipitating from the reaction mixture.

  The composition of the two-phase system obtained by the extraction method of the present invention greatly affects the partition coefficient of the peptide and the polar aprotic solvent between the organic layer and the aqueous layer. In the following, the ratio is expressed as a volume / volume ratio.

  The volume ratio of polar aprotic solvent: toluene is preferably in the range of 1:20 to 1: 2. The volume ratio is preferably in the range of 1:10 to 1: 2, particularly preferably in the range of 1: 6 to 1: 3.

  It has been confirmed that the solubility of the peptide in the combination of toluene and organic solvent 1 is higher than that in toluene alone. Therefore, when the amount of the organic solvent 1 used is sufficiently large, the solubility of the peptide in the organic layer obtained in the extraction method of the present invention is particularly high. The volume ratio of polar aprotic solvent: organic solvent 1 is preferably in the range of 1: 5 to 30: 1. This volume ratio is preferably in the range of 1: 3 to 10: 1, particularly preferably in the range of 1: 1 to 4: 1.

  The volume ratio of toluene: organic solvent 1 is preferably in the range of 50: 1 to 1: 1. This volume ratio is preferably in the range of 20: 1 to 2: 1, particularly preferably in the range of 10: 1 to 2: 1.

  The volume ratio of polar aprotic solvent: water greatly affects the efficiency of the extraction method of the present invention and the solubility of the peptide in the aqueous layer. Specifically, when the volume ratio of polar aprotic solvent: water in the two-phase system is higher than 1: 2, that is, when the amount of polar aprotic solvent contained in the aqueous layer is more than 34% by volume, the aqueous layer The solubility of the peptide in is considerably higher. Accordingly, the volume ratio of polar aprotic solvent: water is preferably in the range of 1:20 to 1: 2. This volume ratio is preferably in the range of 1:10 to 1: 3, particularly preferably in the range of 1: 5 to 1: 3.

  The polar aprotic solvent in the mixture comprising the peptide is preferably selected from the group consisting of DMF and NMP.

  Therefore, both toluene alone and toluene combined with organic solvent 1 are particularly suitable for the peptide extraction method of the present invention. Toluene is an easily reusable and inexpensive solvent and is relatively toxic to humans and aquatic organisms. Therefore, the present invention can be used advantageously on an industrial scale.

  Organic solvent 1 is a group consisting of n-heptane, 2-methyltetrahydrofuran, ethyl acetate (EtOAc), isopropyl acetate, acetonitrile (ACN) and tetrahydrofuran (THF), more preferably a group consisting of EtOAc, isopropyl acetate, ACN and THF In particular, when selected from the group consisting of ACN and THF, the solubility of the peptide in the combination of toluene and organic solvent 1 is particularly high. In a particularly preferred embodiment of the peptide extraction method of the invention, the organic solvent 1 is selected from the group consisting of ACN and THF.

  Component a2) used in the peptide extraction method of the present invention may consist only of water. However, when component a2) further comprises at least one inorganic salt, the miscibility of component a2) with toluene and the miscibility of component a2) with organic solvent 1, ie the solubility of the peptide in the aqueous layer Can be greatly reduced. Furthermore, when component a2) contains at least one inorganic salt, the water content in the organic layer is reduced.

  In a preferred embodiment, component a2) comprises at least one inorganic salt selected from the group consisting of sodium chloride, sodium hydrogen sulfate, potassium hydrogen sulfate, sodium hydrogen carbonate and sodium hydrogen phosphate. In another embodiment, component a2) further comprises other compounds such as acids.

  Specifically, component a2) may contain inorganic salts that do not act as buffers in the range of pH 2-11. When such inorganic salts are added, the solubility of the peptide in the aqueous layer can be reduced, and the time required for phase separation in the extraction method of the present invention can be shortened. For example, component a2) may contain sodium chloride or sodium sulfate. The concentration of inorganic salts contained in component a2) is preferably in the range of 1 to 20% by weight, more preferably in the range of 5 to 15% by weight. Salts such as sodium chloride are used to promote two-phase separation, and salts that act as buffers are used to selectively extract acids or bases in the aqueous layer.

  The pH of component a2) has a great influence on the solubility of the peptide in the aqueous layer and the solubility of some impurities. Furthermore, the pH of component a2) is selected according to the chemical stability of the peptide and the chemical stability of its protecting group (PG). In the extraction method of the present invention, the pH of component a2) is preferably in the range of 2-11 so that most of the tertiary base used in the peptide coupling reaction is contained in the aqueous layer. A range of 8 is particularly preferred. The pH of component a2) can be adjusted by adding acids or bases and / or by using buffers.

  The acid that can be used for adjusting the pH of component a2) is not particularly limited as long as the acid contained in component a2) does not inhibit the peptide extraction method of the present invention and does not cause peptide degradation. You can choose freely. For example, Bronsted acids such as sulfuric acid, hydrochloric acid, phosphoric acid, trifluoroacetic acid and citric acid can be used for this purpose.

  The base that can be used for adjusting the pH of component a2) is not particularly limited as long as the base contained in component a2) does not inhibit the peptide extraction method of the present invention and does not cause degradation of the peptide. You can choose freely. For example, alkali metal hydroxides such as sodium hydroxide, potassium hydroxide and lithium hydroxide are suitable for adjusting the pH of component a2).

  In order to maintain the pH of the aqueous layer within a desired range in the extraction method of the present invention, the component a2) preferably contains a buffer. The buffer is preferably selected from the group consisting of ammonium chloride, sodium hydrogen sulfate, potassium hydrogen sulfate, sodium hydrogen carbonate, sodium carbonate, sodium hydrogen phosphate, sodium dihydrogen phosphate and sodium phosphate. The concentration of the buffer contained in component a2) is preferably in the range of 1 to 10% by weight, and more preferably in the range of 3 to 8% by weight.

  If necessary, the obtained organic layer containing the peptide may be further washed with an aqueous solution at least once. The pH of the aqueous solution used for this purpose is preferably in the range of 2-11.

  Depending on the conditions of the peptide coupling reaction and the reagents used, the organic layer may be a compound having a free primary, secondary or tertiary amino group, such as a peptide having an unprotected N-terminal amino group or A tertiary base may be included as an impurity. In such a case, it is preferable to wash the organic layer with an aqueous solution of pH 2-7.

  The organic layer may also contain a compound having a free carboxylic acid group, such as a peptide having an unprotected C-terminal carboxylic acid group. In such a case, it is preferable to wash the organic layer with an aqueous solution having a pH of 7 to 11.

  The preferred temperature for carrying out the peptide extraction method of the present invention (hereinafter referred to as “extraction temperature”) depends on the choice of the solvent used and the characteristics of the peptide. The extraction temperature greatly affects the miscibility of the solvent used and the solubility of the peptide in the organic and aqueous layers. Therefore, the extraction temperature is selected so that a two-phase system is formed in the extraction method of the present invention and the solubility of the peptide in the organic layer is sufficiently high. The peptide extraction method of the present invention is preferably performed at an extraction temperature of 0 to 60 ° C. The extraction temperature is particularly preferably in the range of 20-30 ° C.

  Depending on the conditions of the peptide coupling reaction and the coupling reagent used, solids may be formed before and / or during the extraction method of the present invention. This is the case, for example, when carbodiimide is used as a coupling reagent. This may require a two-phase filtration obtained by mixing the peptide-containing mixture, polar aprotic solvent, toluene, (optional organic solvent 1), and component a2). Accordingly, in one embodiment of the present invention, a two-phase filtration is performed before separating the organic layer containing the peptide.

  The peptide extracted by the extraction method of the present invention may be any peptide. The peptide extracted by the extraction method of the present invention preferably contains 100 amino acid residues or less, more preferably contains 50 amino acid residues or less, and most preferably contains 20 amino acid residues or less. preferable. The amino acids of the peptide may be D-α-amino acids and / or L-α-amino acids, β-amino acids, at least one primary and / or secondary amino group and at least one carboxylic acid. Other organic compounds containing an acid group may be used. The amino acid is preferably an α-amino acid, more preferably an L-α-amino acid, and among these, an amino acid constituting a protein is particularly preferable.

Peptide Preparation Another aspect of the present invention is a method for preparing a peptide in a liquid phase comprising:
aa) Polar aprotic selected from the group consisting of N, N-dimethylformamide, N, N-dimethylacetamide and N-methyl-2-pyrrolidone in the presence of a coupling reagent and optionally a tertiary base Performing a peptide coupling reaction in an acidic solvent;
bb) extracting the obtained peptide by the above method; and cc) evaporating at least part of the organic layer obtained in step bb).

  As starting material for the peptide coupling reaction of step aa), a combination of two partially protected amino acids, a combination of two partially protected peptides, or a partially protected amino acid and one A single protected peptide combination is used.

  The liquid phase peptide preparation method according to the present invention is very suitable for liquid phase peptide synthesis (LPPS). In one embodiment of the invention, a combination of two peptides prepared by SPPS and partially protected is used in the peptide coupling reaction of step aa). Therefore, the method of the present invention can couple peptide fragments and can be used in combination with SPPS.

  The peptide coupling reaction in step aa) is performed using conventional reaction conditions and reagents usually used for peptide coupling reactions.

  Peptide coupling reactions are conventionally performed by using one or more coupling reagents in a polar aprotic solvent, preferably in the presence of one or more coupling additives, preferably one or more thirds. Performed in the presence of a secondary base.

  Coupling reagents used for peptide coupling reactions are those that do not react with polar aprotic solvents under peptide coupling reaction conditions and substantially undergo epimerization of the stereocenter adjacent to the active carboxylic acid group. The one that does not exist is selected. Therefore, as the coupling reagent, O-1H-benzotriazole phosphonium salt or uronium salt and carbodiimide coupling reagent are preferable.

  The phosphonium salts and uronium salts are BOP (benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphate), PyBOP (benzotriazol-1-yl-oxy-trispyrrolidinophosphonium hexafluoro). Phosphate), HBTU (O- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate), HCTU (O- (1H-6-chloro-benzotriazole) -1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate), TCTU (O- (1H-6-chlorobenzotriazol-1-yl) -1,1,3,3- Tetramethyluronium tetrafluoroborate), HATU (O- (7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophos ), TATU (O- (7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate), TBTU (O- (benzotriazol-1-yl)- 1,1,3,3-tetramethyluronium tetrafluoroborate), TOTU (O- [cyano (ethoxycarbonyl) methyleneamino] -1,1,3,3-tetramethyluronium tetrafluoroborate), HAPyU (O- (benzotriazol-1-yl) oxybis- (pyrrolidino) -uronium hexafluorophosphate), PyAOP (benzotriazol-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate), COMU ( 1-[(1- (Cyano-2-ethoxy-2-oxoethylideneaminooxy) -dimethylamino-morpholinomethylene)]-methanaminium hexafluoro Phosphate), PyClock (6-chloro-benzotriazol-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate), PyOxP (O-[(1-cyano-2-ethoxy-2-oxoethylidene) amino] -Oxytri (pyrrolidin-1-yl) -phosphonium hexafluorophosphate), and PyOxB (O-[(1-cyano-2-ethoxy-2-oxoethylidene) amino] -oxytri (pyrrolidin-1-yl) -phosphonium It is preferably selected from the group consisting of (tetrafluoroborate).

  Preferred coupling reagents selected from phosphonium coupling reagents or uronium coupling reagents are TBTU, TOTU and PyBOP.

  Carbodiimide coupling reagents are from diisopropyl-carbodiimide (DIC), dicyclohexyl-carbodiimide (DCC), and water-soluble carbodiimide (WSCDI) (such as 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC), etc.) Is preferably selected from the group consisting of

  Water-soluble carbodiimide is particularly preferable as a carbodiimide coupling reagent, and EDC is most preferable among water-soluble carbodiimides.

  The tertiary base used in the peptide coupling reaction is preferably compatible with the peptide and the coupling reagent, and preferably does not act as a surfactant that inhibits the extraction method of the present invention.

The pKa of the tertiary base conjugate acid used in the peptide coupling reaction is preferably 7.5 to 15, and more preferably 7.5 to 10. Tertiary bases include trialkylamines such as N, N-diisopropylethylamine (DIPEA) and triethylamine (TEA), N, N-di-C _1-4 alkylanilines such as N, N-diethylaniline, collidine (2 2,4,6-tri-C _1-4 alkyl pyridine such as 4,4,6-trimethylpyridine) and N—C _1-4 alkyl morpholine such as N-methylmorpholine (C _1-4 alkyl) are independent of each other. Are preferably selected from the group consisting of linear or branched C _1-4 alkyl, which may be the same or different. As the tertiary base used in the peptide coupling reaction, DIPEA, TEA and N-methylmorpholine are particularly preferable.

  The coupling additive is preferably a nucleophilic hydroxy compound that forms an activated ester, which is an acidic nucleophilic N-hydroxy group (N is an imide, or an N-acyl or N-aryl substituted triazeno). The triazeno-type coupling additive is preferably an N-hydroxybenzotriazole derivative (or 1-hydroxybenzotriazole derivative) or an N-hydroxybenzotriazine derivative. Such coupling additives are described in WO94 / 07910 and EP 0 410 182.

  Preferred coupling additives are N-hydroxysuccinimide (HOSu), 6-chloro-1-hydroxybenzotriazole (Cl-HOBt), N-hydroxy-3,4-dihydro-4-oxo-1,2,3- Selected from the group consisting of benzotriazine (HOOBt), 1-hydroxy-7-azabenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt) and ethyl-2-cyano-2-hydroxyiminoacetate (CHA) . CHA is available under the trade name OXYMAPURE®. CHA has been found to be an effective coupling additive because it more suppresses the epimerization of the stereocenter of the active carboxylic acid compared to benzotriazole-based coupling additives. In addition, CHA is easier to handle because it is less explosive than, for example, HOBt or Cl-HOBt, and has the additional advantage that the progress of the coupling reaction can be visually monitored by the color change of the reaction mixture. . It is preferred to use HOBt as a coupling additive in the peptide coupling reaction.

  In a preferred embodiment of the invention, the combination of reagents in the peptide coupling reaction is selected from the group consisting of TBTU / HOBt / DIPEA, PyBOP / TEA, EDC / HOBt and EDC / HOBt / DIPEA.

  The reaction solvent for the peptide coupling reaction is selected from the group consisting of DMF, DMA, NMP, and mixtures thereof. Particularly preferred reaction solvents for use in the peptide coupling reaction are selected from the group consisting of DMF and NMP.

  The reaction solvent is preferably substantially free of water. The water content in the reaction solvent is preferably less than 1% by weight, more preferably less than 0.1% by weight, even more preferably less than 0.01% by weight, and 0.001% by weight. It is particularly preferred that it is less than%. The water content in the solvent can be determined by Karl Fischer titration according to standard test method ASTM E203-8 known in the prior art.

  It is preferable that the reaction solvent for the peptide coupling reaction does not substantially contain impurities selected from the group consisting of primary amines, secondary amines, carboxylic acids and aliphatic alcohols. In any starting material used in substoichiometric or stoichiometric amounts, the peptide coupling reaction is less than 1 mol% that causes an undesirable reaction with these impurities during the peptide coupling reaction. The reaction solvent is considered to be substantially free of these impurities.

  The appropriate reaction temperature is selected depending on the coupling reagent used and the stability of the peptide. The reaction temperature for performing the peptide coupling reaction is preferably -15 ° C to 50 ° C, more preferably -10 ° C to 30 ° C, and further preferably 0 ° C to 25 ° C.

  The peptide coupling reaction is preferably performed at atmospheric pressure. However, it is also possible to carry out the peptide coupling reaction under pressures above or slightly below atmospheric pressure.

  The peptide coupling reaction is preferably performed in an ambient atmosphere. However, a protective gas atmosphere such as nitrogen and argon is also preferable.

  As used herein, “reaction time” refers to the time required for the conversion by reaction to be substantially complete. If the amount of starting material used in substoichiometric or stoichiometric amounts is reduced to less than 5 mol% of the initial amount, preferably less than 2 mol% of the initial amount, the conversion by reaction is substantially complete. Is considered. The progress of the reaction can be monitored using analytical methods known in the art, such as high performance liquid chromatography (HPLC) analysis, thin layer chromatography (TLC), mass spectrometry (MS) or HPLC-MS. HPLC is particularly preferred for this purpose.

  The reaction time of the peptide coupling reaction is preferably in the range of 15 minutes to 20 hours, more preferably in the range of 30 minutes to 5 hours, and still more preferably in the range of 30 minutes to 2 hours.

  “Part” in the description of the reaction conditions for the peptide coupling reaction means the ratio of the peptide and / or amino acid used as the starting material for the peptide coupling reaction to the total weight. The amount of reaction solvent used is preferably 1-30 parts, more preferably 5-10 parts.

  The amount of the coupling reagent used is preferably 0.9 to 5 molar equivalents, more preferably 1 to 1.5 molar equivalents with respect to the reactive C-terminal carboxylic acid group.

  0.1-5 molar equivalent is preferable with respect to a coupling reagent, and, as for the usage-amount of a coupling additive, 0.5-1.5 molar equivalent is more preferable.

  1-10 molar equivalent is preferable with respect to a coupling reagent, and, as for the usage-amount of a tertiary base, 2-3 molar equivalent is more preferable.

  Any peptide can be obtained by the liquid phase peptide preparation method of the present invention.

  The peptide obtained by the liquid phase peptide preparation method of the present invention preferably contains 100 amino acid residues or less, more preferably contains 50 amino acid residues or less, and contains 20 amino acid residues or less. Most preferred. The amino acid of the peptide may be a D-α-amino acid, an L-α-amino acid, or a β-amino acid, at least one primary and / or secondary amino group and at least one carboxylic acid Other organic compounds containing a group may be used. The amino acid of the peptide obtained by the liquid phase peptide preparation method of the present invention is preferably an α-amino acid, more preferably an L-α-amino acid, and among these, an amino acid constituting a protein is particularly preferable.

  After the extraction step, the organic layer containing the peptide is preferably partially evaporated. Therefore, in the present application, the organic layer thus obtained is referred to as a “partially evaporated organic layer”. The temperature at which the partial evaporation is performed is not particularly limited, and is selected depending on the thermal stability of the peptide and the characteristics of toluene or the characteristics of the mixture of toluene and organic solvent 1. The partial evaporation of the organic layer is preferably performed at a temperature of 30 to 50 ° C. If necessary, the partial evaporation of the organic layer is carried out under reduced pressure of 20 to 1000 mbar (20 to 1000 hPa), preferably under reduced pressure of 50 to 200 mbar (50 to 200 hPa). One skilled in the art will appreciate that the pressure at which the organic layer is partially evaporated is preferably adjusted according to the desired temperature at which the evaporation operation is performed.

  Since toluene and organic solvent 1 have sufficient volatility, partial evaporation of the organic layer containing the peptide can be easily performed.

  In one embodiment of the present invention, the organic layer containing the peptide is directly evaporated to dryness and the remaining residue is dissolved in a solvent other than toluene or organic solvent 1. However, when the organic layer containing the peptide contains more than 30% by volume of a solvent selected from the group consisting of MeTHF and THF, it is preferable not to evaporate and dry completely for safety reasons. Instead, it is possible to evaporate to dryness after partially evaporating the organic layer containing the peptide and adding toluene.

  Since toluene contained in the organic layer forms an azeotrope with water, water present in a trace amount in the organic layer containing the peptide is effectively removed in the step of partially evaporating the organic layer.

  In a preferred embodiment, the majority of the peptide is precipitated by mixing the partially evaporated organic layer and the organic solvent 2.

  In another preferred embodiment of the invention, the organic layer containing the peptide is evaporated to dryness and the remaining residue is dissolved in a solvent that is neither toluene nor organic solvent 1. The resulting solution is then mixed with the organic solvent 2 to precipitate the peptide.

  The volume ratio of partially evaporated organic layer: organic solvent 2 used to precipitate the peptide has a significant effect on the integrity of the precipitation process and the properties of the precipitated peptide. In the following, the ratio is expressed as a volume / volume ratio.

  The volume ratio of the partially evaporated organic layer: organic solvent 2 is preferably in the range of 1:20 to 1: 1. This volume ratio is preferably in the range of 1:12 to 1: 2, particularly preferably in the range of 1: 6 to 1: 3.

  The organic solvent 2 is preferably selected from organic solvents having a boiling point of less than 160 ° C. at atmospheric pressure. The solubility of the peptide in the organic solvent 2 is preferably lower than the solubility in toluene and / or the solubility in a mixture of toluene and the organic solvent 1. The organic solvent 2 is preferably selected from the group consisting of acetonitrile, diethyl ether, diisopropyl ether and n-heptane, more preferably selected from the group consisting of acetonitrile, diethyl ether and diisopropyl ether, diisopropyl ether and n It is particularly preferred that it is selected from the group consisting of heptane.

  Since the partially evaporated organic layer containing peptide is substantially free of polar aprotic solvent, the amount of organic solvent 2 required for peptide precipitation is a crude reaction obtained by peptide coupling reaction. Significantly less than the amount used in conventional precipitation processes using mixtures. Furthermore, unlike the conventional precipitation process, the peptide precipitates as a non-sticky solid.

  In the precipitation step, it is preferred that at least 80% by weight of the peptide contained in the partially evaporated organic layer is precipitated as a solid. More preferably, at least 90% by weight of the peptide contained in the partially evaporated organic layer precipitates as a solid. Even more preferably, at least 95% by weight of the peptide contained in the partially evaporated organic layer precipitates as a solid. It is particularly preferred that at least 98% by weight of the peptide contained in the partially evaporated organic layer is precipitated as a solid.

  The temperature at which the precipitation step is performed (this temperature is hereinafter referred to as “precipitation temperature”) depends on the composition of the partially evaporated organic layer, the choice of the organic solvent 2 and the properties of the peptide.

  The precipitation temperature has a great influence on the integrity of the precipitation of the peptide and the physical properties of the precipitated peptide. The precipitation step is preferably performed at a precipitation temperature of −10 to 60 ° C., and more preferably performed at a precipitation temperature of −10 to 30 ° C. However, the precipitation temperature is particularly preferably in the range of −10 to 0 ° C.

  Since the partially evaporated organic layer containing the peptide is substantially free of polar aprotic solvent, the precipitated peptide can be easily filtered off. Thus, the time required for the filtration process is greatly reduced. The precipitated peptide is preferably filtered off and dried under reduced pressure.

  However, it is also possible to separate the precipitated peptides by centrifugation.

  If desired, the filtrate recovered by filtration can be partially evaporated again before being subjected to a precipitation step, and thus a second batch of peptide precipitate can be recovered.

  In another embodiment of the invention, the partially evaporated organic layer containing the peptide is treated directly with a reagent that cleaves one or more protecting groups (PG) of the peptide. Since the partially evaporated organic layer containing the peptide is substantially free of polar aprotic solvent, the reagent for cleaving one or more PGs of the peptide is not particularly limited and can be freely selected. . For example, a partially evaporated organic layer containing a peptide can be treated with an acid decomposing reagent, in which an undesirable reaction between the acid decomposing reagent and a polar aprotic solvent can also result in cleavage of the protecting group. There is no suppression. This embodiment of the invention is particularly preferred when the N-terminal PG of the peptide is a tert-butoxycarbonyl (Boc) group.

  In another embodiment of the invention, the partially evaporated organic layer is used to perform other reactions such as disulfide bridge formation.

  In another embodiment of the invention, a reagent that cleaves one or more PGs of a peptide is added directly to the reaction mixture obtained by the peptide coupling reaction. After completion of cleavage of the targeted PG, the resulting peptide is extracted from the reaction mixture. This embodiment of the invention is particularly suitable when the N-terminal PG of the peptide is a fluorenyl-9-methoxycarbonyl (Fmoc) group.

  In one particular embodiment, the peptide after PG cleavage is extracted with toluene or a mixture of toluene and organic solvent 1. This is usually done for Fmoc protected peptides that are difficult to keep in a solution that does not contain NMP or DMF. After cleavage of Fmoc, such peptides can be extracted into an organic layer containing toluene and optionally organic solvent 1.

  In the case of peptides protected with Boc, contrary to the above, NMP and DMF must be removed before cleaving Boc. Such peptides are usually dissolved in toluene, ethyl acetate or heptane in the presence of> 5% by volume TFA.

  In yet another embodiment of the invention, the organic layer comprising the peptide is evaporated to dryness as described above, the remaining residue is dissolved in a solvent other than toluene or organic solvent 1, and then one or more PGs of the peptide Add reagent to cleave.

Protecting group The protecting group (PG) used in the present invention for protecting the side chain functional group of an amino acid or peptide, or protecting the N-terminal amino group or C-terminal carboxylic acid group of an amino acid or peptide includes the following four groups: Classified into different groups.
1. PG that can be cleaved under basic cleavage conditions (hereinafter referred to as “basic PG”)
2. PG that can be cleaved under strongly acidic cutting conditions but cannot be cleaved under weakly acidic cutting conditions (hereinafter referred to as “strong PG”)
3. PG that can be cut under mildly acidic cutting conditions (hereinafter referred to as “weak PG”)
4). PG that can be cleaved under reducing conditions (hereinafter referred to as "reduced PG")
5. PG cleavable under saponifiable cutting conditions (hereinafter referred to as “saponified PG”)

  In the liquid phase peptide preparation method of the present invention, PG used according to the conventional method, and typical reaction conditions, parameters and reagents for cleaving PG are known in the art, for example, TW Greene, PGM Wuts “Greene's Protective Groups in Organic Synthesis” John Wiley & Sons, Inc., 2006; or P. Lloyd-Williams, F. Albericio, E. Giralt, “Chemical Approaches to the Synthesis of Peptides and Proteins” CRC: Boca Raton, Florida, 1997.

  Basic cleavage conditions include treatment of the peptide with a basic cleavage solution. The basic cleavage solution is preferably composed of a basic reagent and a solvent. The basic reagent used in the present invention is preferably a secondary amine, such as diethylamine (DEA), piperidine, 4- (aminomethyl) piperidine, tris (2-aminoethyl) amine (TAEA), morpholine, dicyclohexylamine, 1 More preferably, it is selected from the group consisting of 1,3-cyclohexanebis (methylamine) -piperazine, 1,8-diazabicyclo [5.4.0] undec-7-ene and mixtures thereof. Even more preferably, the basic reagent used in the method for preparing a liquid phase peptide of the present invention is selected from the group consisting of DEA, TAEA and piperidine.

  The basic cleavage solution may further comprise additives, which are 6-chloro-1-hydroxy-benzotriazole, 1-hydroxy-7-azabenzotriazole, 1-hydroxybenzotriazole, ethyl-2-cyano. It is preferably selected from the group consisting of -2-hydroxyiminoacetate and mixtures thereof.

  The solvent of the basic cleavage solution is preferably the same as the polar aprotic solvent used in the peptide coupling reaction. Accordingly, the solvent of the basic cleavage solution is preferably selected from the group consisting of DMF, DMA and NMP. Alternatively, the peptide-containing organic layer obtained by the method of extracting the peptide from the reaction mixture after the peptide coupling reaction is evaporated to dryness as described above, and the remaining residue is DMF, DMA, pyridine, NMP, It can also be dissolved in any one of the solvents selected from the group consisting of acetonitrile and mixtures thereof and then treated with a basic cleavage solution. As described in Example 1, DMF or NMP may be required to retain the peptide in the reaction mixture solution of the Fmoc cleavage reaction.

  In the description of basic, strongly acidic, weakly acidic, or reducing cleavage conditions, “parts” and “% by weight” mean a ratio to the weight of peptides having a group of PG corresponding to each cleavage condition. For example, “use 5 parts of basic cleavage solution” means that 5 g of basic cleavage solution is used to treat 1 g of peptide having base type PG.

  The amount of basic cleavage solution used is preferably 5 to 20 parts, more preferably 5 to 15 parts. The amount of the basic reagent is preferably in the range of 1 to 30% by weight, more preferably in the range of 10 to 25% by weight, more preferably in the range of 15 to 20% by weight with respect to the total weight of the basic cleavage solution. Even more preferred is a range.

  As defined in the present invention, strongly acidic cleavage conditions include treatment of the peptide with a strongly acidic cleavage solution. The strongly acidic cleavage solution contains an acid degradation reagent. Acid decomposing reagents include TFA, hydrochloric acid (HCl), hydrochloric acid (HCl) aqueous solution, hydrofluoric acid (HF) solution and Bronsted acids such as trifluoromethanesulfonic acid; trifluoroborate-diethyl ether adduct and trimethylsilyl bromide Preferably selected from the group consisting of Lewis acids; and mixtures thereof.

  The strongly acidic cleavage solution is one or more selected from the group consisting of dithiothreitol, ethanedithiol, dimethyl sulfide, triisopropylsilane, triethylsilane, 1,3-dimethoxybenzene, phenol, anisole, p-cresol, and mixtures thereof. It is preferable to contain the capture agent. The strongly acidic cleavage solution may further comprise water, a solvent or a mixture thereof, which is a solvent that is stable under strong cleavage conditions.

  The solvent of the strongly acidic cleavage solution is preferably the same as the solvent contained in the partially evaporated organic layer containing the peptide. Therefore, the solvent of the strongly acidic cleavage solution is toluene or a combination of toluene and organic solvent 1. Alternatively, the organic layer containing the peptide is evaporated to dryness as described above and the remaining residue is dissolved in any one of the solvents selected from the group consisting of ACN, toluene, DCM, TFA, and mixtures thereof. You can also. Since toluene and organic solvent 1 have sufficient volatility, the organic layer can be easily evaporated.

  The amount of the strongly acidic cleavage solution used is preferably 10 to 30 parts, more preferably 15 to 25 parts, and even more preferably 19 to 21 parts. The amount of the acid decomposing reagent is preferably in the range of 30 to 350% by weight, more preferably in the range of 50 to 300% by weight, and in the range of 70 to 250% by weight with respect to the total weight of the strongly acidic cleavage solution. It is even more preferable that it is in the range of 100 to 200% by weight. The total amount of scavenger used is preferably 1 to 25% by weight, more preferably 5 to 15% by weight, based on the total weight of the strongly acidic cleavage solution.

  The weakly acidic cleavage conditions according to the present invention include treatment of the peptide with a weakly acidic cleavage solution. The weakly acidic cleavage solution contains an acid degradation reagent. The acid decomposing reagent is preferably selected from the group consisting of Bronsted acids such as TFA, trifluoroethanol, hydrochloric acid (HCl), acetic acid (AcOH), mixtures thereof, and / or aqueous mixtures thereof.

  The weakly acidic cleavage solution may further comprise water, a solvent or a mixture thereof, which is a solvent that is stable under weak cleavage conditions. The solvent of the weakly acidic cleavage solution is preferably the same as the solvent contained in the partially evaporated organic layer containing the peptide. Therefore, the solvent of the weakly acidic cleavage solution is toluene or a combination of toluene and organic solvent 1. Alternatively, the organic layer containing the peptide is evaporated to dryness as described above and the remaining residue is dissolved in any one of the solvents selected from the group consisting of ACN, toluene, DCM, TFA, and mixtures thereof. You can also

  The amount of the weakly acidic cleavage solution used is preferably 4 to 20 parts, and more preferably 5 to 10 parts. The amount of the acid decomposing reagent is preferably in the range of 0.01 to 5% by weight, more preferably in the range of 0.1 to 5% by weight, based on the total weight of the weakly acidic cleavage solution. Even more preferably, it is in the range of 15 to 3% by weight.

  The reducing cleavage conditions used in one embodiment of the present invention include treatment of the peptide with a reducing cleavage mixture. The reducing cleavage mixture includes a catalyst, a reducing agent and a solvent.

The catalyst used for the reductive cleavage conditions is selected from the group consisting of Pd (0) derivatives, Pd (II) derivatives and metal palladium-containing catalysts, Pd [PPh ₃ ] ₄ , PdCl ₂ [PPh ₃ ] ₂ , more preferably selected from the group consisting of Pd (OAc) ₂ and palladium-carbon (Pd / C). Pd / C is particularly preferred.

Reducing agents are Bu ₄ N ⁺ BH ₄ ⁻ ; NH ₃ BH ₃ ; Me ₂ NHBH ₃ ; tBu-NH ₂ BH ₃ ; Me ₃ NBH ₃ ; HCOOH / DIPEA; PhSO ₂ H, tolSO ₂ Na or i-BuSO ₂ It is preferably selected from the group consisting of sulfinic acid containing Na; mixtures thereof; and hydrogen molecules, more preferably tolSO ₂ Na or hydrogen molecules.

  The solvent used under reducing cleavage conditions is preferably the same as the solvent contained in the partially evaporated organic layer containing the peptide. Therefore, the solvent used under reducing cleavage conditions is preferably toluene or a combination of toluene and organic solvent 1. Alternatively, the organic layer containing the peptide is evaporated to dryness as described above, and the remaining residue is any one of the solvents selected from the group consisting of NMP, DMF, DMA, pyridine, ACN and mixtures thereof, More preferably, it can be dissolved in NMP, DMF or a mixture thereof. Peptides are preferably soluble in these solvents used under reducing cleavage conditions and are preferably dissolved in such solvents.

  The amount of reducing cutting solution used is preferably 4 to 20 parts, more preferably 5 to 10 parts.

  Saponifiable cleavage conditions include treatment of the peptide with a saponifiable cleavage solution. The saponifiable cleavage solution preferably comprises a saponification reagent and a solvent. The saponification reagent used in the present invention is preferably an alkali metal hydroxide or an alkaline earth metal hydroxide, and is selected from the group consisting of sodium hydroxide, lithium hydroxide and potassium hydroxide. Is more preferable. Even more preferably, the saponification reagent used in the liquid phase peptide preparation method of the present invention is sodium hydroxide.

  The solvent of the saponifiable cleavage solution preferably contains a mixture of a solvent selected from the group consisting of THF, MeTHF, ethanol, methanol and dioxane and water.

  According to the present invention, base type PG cannot be cleaved under strongly acidic cleavage conditions and weakly acidic cleavage conditions. It is preferable that the basic PG cannot be cleaved under strongly acidic cleavage conditions, weak cleavage conditions, and reducing cleavage conditions.

  “Strong PG” is understood to be a protecting group that cannot be cleaved under mildly acidic and basic cleavage conditions. It is preferable that the strong PG cannot be cleaved under mildly acidic cleavage conditions, basic cleavage conditions, and reducing cleavage conditions. Strongly acidic PGs such as Bzl are usually cleaved by hydrogenation. Normally, comprehensive deprotection of peptides is performed by hydrogenation under very mild conditions.

  Weak PG cannot be cleaved under basic cleavage conditions, but can be cleaved under strongly acidic cleavage conditions. Preferably, the weak PG cannot be cleaved under basic or reducing cleavage conditions, but can be cleaved under strongly acidic cleavage conditions.

  According to one embodiment of the present invention, the base PG is preferably Fmoc. The strong PG is preferably selected from the group consisting of Boc, tBu, OtBu and Cbz. The weak PG is preferably selected from the group consisting of Trt and a 2-chlorophenyldiphenylmethyl group. The reduced PG is preferably selected from the group consisting of Bzl, N-methyl-9H-xanthene-9-amino group and Cbz. The saponified PG is preferably OMe.

  In the liquid phase peptide preparation method of the present invention, before the next peptide coupling reaction, the N-terminal PG of the peptide is removed by a deprotection reaction. According to the invention, the N-terminal PG is preferably Fmoc or Boc.

  In one embodiment of the invention, Fmoc is highly preferred as the N-terminal PG in LPPS because it can be easily removed under basic conditions. Furthermore, the use of Fmoc as the N-terminal PG of the peptide is compatible with the side chain PG in the orthogonal system. The “orthogonal system” is defined by G. Baranay and R. B. Merrifield (JACS, 1977, 99, 22, pp. 7363-7365).

  In yet another embodiment of the invention, Boc is highly preferred as the N-terminal PG of peptides used in the liquid phase peptide preparation method. The removal can be performed under strongly acidic conditions. Furthermore, when Boc is used as the N-terminal PG, it is compatible with the side chain PG in the orthogonal system.

  According to the present invention, the C-terminal PG of the peptide is removed in the final deprotection step.

As C-terminal PG, OtBu, Blz, OMe, NH 2, 2- chlorophenyl - diphenylmethyl ester or N- methyl -9H- xanthene-9-amides are preferred.

  In one embodiment of the present invention, Bzl can be easily removed under the reductive cleavage conditions described above, and is therefore highly preferred as the C-terminal PG in the liquid phase peptide preparation method. Furthermore, the use of Bzl as the C-terminal PG is compatible with the side chain PG in the orthogonal system.

  In another embodiment of the invention, OtBu is used as the C-terminal PG in the liquid phase peptide preparation method. The removal can be performed under the above-mentioned strongly acidic cleavage conditions. Furthermore, when OtBu is used as the C-terminal PG, it is compatible with the side chain PG in the orthogonal system.

  In another embodiment of the invention, OMe is used as the C-terminal PG of the liquid phase peptide preparation method. OMe can be easily cleaved by saponification and is particularly useful when the N-terminal PG of the peptide is Boc.

  In yet another embodiment of the invention, the solubility of the peptide in the organic layer can be further enhanced by using hydrophobic PG at the C-terminus of the peptide. For this purpose, weak CPG cleavable under mildly acidic conditions may be used to protect the C-terminal carboxylic acid group of the peptide. Examples of such weak PG include 2-chlorophenyldiphenylmethyl ester and N-methyl-9H-xanthene-9-amide. These PGs are particularly useful for the synthesis of peptide fragments, and the obtained peptide fragments can be used for peptide synthesis by the convergent method. These C-terminal carboxylic acid protecting groups (PG) also have another important advantage. That is, since these C-terminal carboxylic acid protecting groups (PG) are cleaved under weakly acidic conditions, a protected peptide used as a peptide fragment in a convergent synthesis method is synthesized using liquid phase synthesis instead of SPPS. be able to. Indeed, 2-chlorophenyldiphenylmethyl ester and N-methyl-9H-xanthene-9-amide are chemical functional groups used as linkers for SPPS resins for the purpose of synthesizing protected peptide fragments.

  According to the present invention, undesirable side reactions are avoided by protecting the hydroxy group, amino group, thio group and carboxylic acid group of the amino acid side chain of the peptide obtained by the liquid phase peptide preparation method with an appropriate PG. Is desirable. Furthermore, the use of PG in the side chain usually improves the solubility of the peptide in polar aprotic solvents and the solubility of the peptide in toluene and / or a combination of toluene and organic solvent 1.

  Usually, the side chain PG is selected so as not to be removed during the N-terminal amino group deprotection step in the liquid phase peptide preparation method. Therefore, the PG of the N-terminal amino group or C-terminal carboxylic acid group and the PG of the side chain are usually different, and these PGs are preferably compatible in the orthogonal system.

  According to the invention, preferred side groups are tBu, Trt, Boc, OtBu and Cbz.

  If the amino acid sequence identical to the amino acid sequence of the target peptide is obtained by the liquid phase peptide preparation method, the N-terminal PG, the C-terminal PG and the side chain PG are removed, and the unprotected target peptide is removed. It is preferable to obtain. This process is called “global deprotection”. In the liquid phase peptide preparation method, it is preferable to select a PG that can perform comprehensive deprotection under the above-mentioned weakly acidic, strongly acidic, or reducing cleavage conditions depending on the characteristics of the PG.

  The side chain PG is usually retained until LPPS is complete. Comprehensive deprotection can be performed under conditions applicable to the various side chain PGs used. If different types of side chain PGs are selected, these PGs may be cleaved continuously, for example when synthesizing branched peptides. It is advantageous to select side chain PG that can be cleaved at the same time, and in addition, it is further advantageous to select side chain PG that can also cleave N-terminal PG or C-terminal PG of peptides prepared by LPPS at the same time.

  In one embodiment of the invention, the N-terminal PG of the peptide in the partially evaporated organic layer can be removed directly. Therefore, in this case, it is not necessary to precipitate the peptide using the organic solvent 2, and the LPPS of the present invention can be carried out, for example, as continuous LPPS without isolating the intermediate peptide.

  Depending on the properties of the N-terminal PG of the peptide, suitable cleavage conditions for this step can be selected.

  When the N-terminal PG of the peptide is a strong PG or a weak PG as described above, the organic layer containing the peptide is preferably treated with TFA or HCl. Since the organic layer containing the peptide is substantially free of polar aprotic solvent, removal of the N-terminal PG of the peptide is not inhibited by undesired reactions between TFA or HCl and the polar aprotic solvent. In one embodiment of the invention, the N-terminal PG of the peptide is a Boc group.

  If the N-terminal PG of the peptide is a base PG as described above, the peptide can be deprotected using an organic base as is known in the art. For this purpose, the reaction mixture obtained by the peptide coupling reaction is treated directly with a basic reagent selected from the group consisting of DEA, TAEA and piperidine and then reacted with a peptide having an unprotected N-terminus. Extraction from the mixture is preferred. Alternatively, the organic layer containing the peptide is treated with a basic reagent. As another alternative, the organic layer containing the peptide is evaporated to dryness as described above and the remaining residue is dissolved in one of the solvents selected from the group consisting of DMF, DMA, pyridine, NMP and mixtures thereof. Can then be treated with a basic reagent.

  In a preferred embodiment of the invention, the N-terminal PG of the peptide is a fluorenyl-9-methoxycarbonyl (Fmoc) group. Cleavage of the Fmoc group of the peptide is accompanied by the formation of dibenzofulvene. When DEA or piperidine is used as the basic reagent and the solvent of the basic cleavage solution is acetonitrile, the resulting solution containing the unprotected N-terminal peptide is washed with a hydrocarbon such as n-heptane. This substantially removes dibenzofulvene. When TAEA is used as a basic reagent for cleaving the Fmoc group, the resulting solution is treated with the extraction method of the present invention. Thus, the dibenzofulvene is substantially removed from the solution containing the peptide having an unprotected N-terminus prior to performing a subsequent peptide coupling reaction.

  After cleavage of the N-terminal PG of the peptide, the solution containing the peptide having an unprotected N-terminus can be at least partially evaporated and used for the next peptide coupling reaction or a comprehensive deprotection step.

  Thus, the present invention provides a continuous LPPS method that has many advantages over commonly used SPPS methods.

  In the case of the continuous LPPS of the present invention, the concentration of the reagent in the reaction mixture in the peptide coupling reaction and the deprotection reaction is higher than in the case of SPPS. As a result, the reaction time is shorter and a smaller volume of the batch reactor can be used to synthesize a predetermined amount of the desired peptide. The total time required for peptide synthesis by continuous LPPS of the present invention is approximately the same as the total time required for synthesis using SPPS. Thus, working costs can be saved by using the continuous LPPS of the present invention.

  The excess of amino acids or peptides having an unprotected C-terminal carboxylic acid group (1.1-1.2 equivalents) required in the peptide coupling reaction of LPPS of the present invention is the corresponding SPPS peptide cup. Less than the excess of amino acids or peptides in the ring reaction (1.5 equivalents or more). In addition, SPPS requires a large amount of solvent to rinse the resin after each peptide coupling step. Thus, the amount of solvent required for SPPS is significantly higher than the continuous LPPS of the present invention. Thus, material costs can be saved significantly when using the continuous LPPS of the present invention compared to using SPPS.

  In addition to the above, it is known that the scale-up of the continuous LPPS method of the present invention is easier than the scale-up of the corresponding SPPS method, and the target peptide prepared by the continuous LPPS of the present invention is Higher purity than the same peptide prepared by SPPS.

  In summary, the continuous LPPS of the present invention has many advantages over other peptide synthesis methods known in the prior art and is particularly useful for the preparation of peptides on an industrial scale.

  The following examples explain in detail typical embodiments of the present invention, but the present invention is not limited thereto.

  Unless otherwise stated, all experiments were performed at room temperature (20 ± 3 ° C.) and atmospheric pressure (1013 ± 50 kPa).

Description of method
A) HPLC analysis
Detection in HPLC method A was performed using a UV photodiode array detector.

Step 1 Sample preparation:
Mobile phase A: water containing 0.1% by volume TFA Mobile phase B: ACN containing 0.085% by volume TFA

Step 2 Chromatographic conditions:
MIH-009-2TG11 Column: Purospher Star RP18 55 × 4mm
Oven temperature: 40 ℃
Flow rate: 2.0mL / min Detector wavelength: 215nm
Gradient time: 15 minutes Gradient composition: 2 to 78% B (5 minutes) → 78 to 98% B (10 minutes)

Step 3 Chromatographic profile analysis:
The composition of the isolated product was determined by measuring the area of all peaks in the chromatography. The predicted product purity determined corresponds to the peak area (%) corresponding to the product.

1. Equipment and instruments Gas chromatograph: GC equipped with a flame ionization detector and autoinjector system, connected to data acquisition software
GC column for analysis: fused silica column
Length 50m; Inner diameter 0.53mm; Stationary phase: CP SIL 8CB DF = 5.0μm
Reagent: Methanol (analysis grade)

2. Sample preparation Test solution and control solution
A sample of 400 μL was accurately placed in a 10 mL volumetric flask, and methanol was added up to the marked line.

3. Chromatographic conditions Carrier gas: Helium 30kPa
Oven temperature: 35 ℃ (14 minutes) → (5 ℃ / minute) → 55 ℃ (3 minutes) → (5 ℃ / minute) → 110 ℃ (5 minutes) → (10 ℃ / minute) → 225 ℃ (5 minutes) )
Injector temperature: 225 ° C
Detector temperature: 260 ℃
Injection volume: 1μL
Injection method: Split Split flow rate: 85mL / min Ratio: 24

Measurement of filterability The mixture containing the precipitated peptide was transferred to a 2.7 cm diameter filtration column equipped with a 20 μm pore size filter. Filtration was carried out at 20 ° C. and a pressure of 50 mbar. The flow rate and cake height were measured, and the filtration coefficient K was determined by the following equation.
K = amount of mother liquor (mL) x cake height (cm) / filter area (cm ² ) / pressure (bar) / filtration time (min)

Example 1 Extraction of NMP in MeTHF / THF / NaCl Solution System, EtOAc / THF / NaCl Solution System, and Toluene / THF / NaCl Solution System The extractability of toluene / THF mixed solvent (invention) was measured as MeTHF / THF mixed solvent and EtOAc. Comparison with extractability of / THF mixed solvent (comparative example). The experiment was performed using an aqueous solution containing 150 g / L NaCl. No peptide was included in the system of this example.
The volume ratio was as follows.
NMP: EtOAc: THF: NaCl solution = 1: 3: 3: 3
NMP: MeTHF: THF: NaCl solution = 1: 3: 3: 3
NMP: toluene: THF: NaCl solution = 1: 3: 3: 3
The NMP fraction in the aqueous layer was measured by GC. The experimental results are summarized in Table 1.

  As is clear from Table 1 above, when extraction was performed using a toluene / THF mixed solvent, the NMP fraction in the aqueous layer increased as compared with the case where extraction was performed using EtOAc / THF or MeTHF / THF. Correspondingly, the NMP content in the organic layer after extraction with toluene / THF was less than when extracted with MeTHF / THF or EtOAc / THF.

Example 2 Synthesis of H-Tyr (Bzl) -Leu-OBzl
Example 2.1 LPPS of Boc-Tyr (Bzl) -Leu-OBzl
Boc-Tyr (Bzl) -OH (4.7 g, 12.7 mmol) and H-Leu-OBzl · Tos (5.0 g, 12.7 mmol) were dissolved in DMF (25 mL) at 20 ° C. The reaction mixture was cooled to −8 ° C. and then HOBt · H ₂ O (2.0 g, 13.1 mmol, 1.0 equiv) and EDC · HCl (2.8 g, 14.6 mmol) were added. The reaction temperature was maintained in the range of -5 to -10 ° C until the completion of the reaction was confirmed by HPLC. The progress of the reaction was monitored by diluting 5 μL of the sample obtained from the reaction mixture 50 times with acetic acid: water (9: 1) and analyzing according to the above-mentioned MIH-009-2TG11 method.

Example 2.2 Boc cleavage: H-Tyr (Bzl) -Leu-OBzl
Toluene (90 mL) was added to the mixture prepared according to Example 2.1 and extracted sequentially from the reaction mixture with:
1) Aqueous solution (90mL) containing 20g / L NaCl
2) Aqueous solution (90 mL) containing 150 g / L NaCl and 50 g / L NaHCO ₃
3) Aqueous solution (90 mL) containing 20 g / L NaCl and 50 g / L NaHCO ₃
4) Aqueous solution (90 mL) containing 20 g / L NaCl and 50 g / L NaHCO ₃
5) Aqueous solution (90mL) containing 150g / L NaCl

  The combined organic layers were then concentrated under reduced pressure at 35 ° C. until the volume was 20 mL.

  The Boc protecting group was removed by adding phenol (0.25 g, 2.6 mmol) and TFA (20 mL) at 15 ° C. After confirming the completion of the reaction by HPLC, the reaction mixture was evaporated at 35 ° C. under reduced pressure. Residual TFA was removed by coevaporation with toluene (3 × 25 mL). The progress of the reaction was monitored by diluting a sample of 5 μL obtained from the reaction mixture 30-fold with methanol and analyzing according to the above-mentioned MIH-009-2TG11 method.

Example 3 (Comparative) Effect of residual DMF on removal of Boc protecting group: H-Pro-Ile-Leu-Pro-Glu (OBzl) -Glu (OBzl) -Tyr (Bzl) -Leu-OBzl
Boc-Pro-Ile-Leu-Pro-Pro-OH (3.5g), H-Glu (OBzl) -Glu (OBzl) -Tyr (Bzl) -Leu-OBzl (5.0g) and HOBt (0.88g) in DMF Dissolved in (20 mL). EDC.HCl (1.2 g) and TEA (1.5 mL) were added, and the coupling reaction was performed overnight with stirring at -6 to 0 ° C. Completion of the reaction was confirmed by HPLC (MIH-009-2TG11 method).

The reaction mixture was filtered to remove insoluble salts. 1 mL of a sample obtained from the reaction mixture is mixed with the organic solvent described in Table 2 below, and then extracted with 3 mL of an aqueous solution consisting of NaCl (15% w / v) and Na ₂ CO ₃ (2.5% w / v). It was. The DMF content in the organic layer was determined by GC.

  Extraction with EtOAc (Test No. 2) reduced the DMF content in the organic layer compared to extraction with DCM (Test No. 1).

  The materials obtained in tests No. 1 and 2 were further processed as follows. The organic layer was separated and the solvent was changed by co-evaporation with toluene three times (bath temperature = 40 ° C., pressure = 50 mbar). After complete evaporation of the volatile solvent, toluene (4 mL) and phenol (0.05 g) were added to the evaporation residue. Boc cleavage was performed at 0 ° C. by adding TFA (3.5 mL). This reaction was monitored by HPLC (MIH-009-2TG11 method).

  The results obtained are summarized in Table 3 and shown in the graph of FIG.

Traces of DMF results obtained in the material significantly inhibited the removal of the Boc protecting group. Therefore, the progress of Boc cleavage in the material extracted with DCM was significantly slower than that of the material extracted with EtOAc.

  By using the extraction method of the present invention, a polar aprotic solvent such as DMF can be efficiently separated from the isolated peptide. Therefore, it is expected that the acidolytic cleavage of the peptide material isolated by the extraction method of the present invention will proceed rapidly.

Example 4 (Comparative Example) Coupling of Boc-Ser (OBzl) -OH with H-Phe-Pro-Ile-Leu-Pro-Pro-Glu (OBzl) -Glu (OBzl) -Tyr-Leu (OBzl)
Boc-Ser (OBzl) -OH (1.62 g, 5.5 mmol) was dissolved in DMF (25 mL) at 20 ° C. and the crude product H-Phe-Pro-Ile-Leu-Pro-Pro-Glu (OBzl ) -Glu (OBzl) -Tyr-Leu (OBzl). HOBt · H ₂ O (0.89 g, 5.8 mmol) and EDC · HCl (1.2 g, 6.3 mmol) were added and the reaction mixture was cooled to 5 ° C. The reaction mixture was maintained at this temperature until completion of the reaction was confirmed by HPLC. The progress of the reaction was monitored by diluting 5 μL of the sample obtained from the reaction mixture 50 times with acetic acid: water (9: 1) and analyzing according to the above-mentioned MIH-009-2TG11 method.

a) Extraction with MeTHF and precipitation in DIPE
25 mL of a reaction mixture containing 5 g of Boc-Ser (Bzl) -Phe-Pro-Ile-Leu-Pro-Pro-Glu (OBzl) -Glu (OBzl) -Tyr (Bzl) -Leu (OBzl), 100 g / L NaCl Mixed with an aqueous solution containing (75 mL) and MeTHF (75 mL). After thoroughly mixing and phase separation (about 4 minutes), the lower aqueous layer was removed. Furthermore, extraction from the upper organic layer was performed three times using an aqueous solution (3 × 75 mL) containing 100 g / L NaCl. The organic layer was then isolated and partially evaporated at 30 ° C. and 60 mbar until the remaining volume was 10 mL. The peptide was precipitated by dropwise addition of the partially evaporated organic layer while stirring DIPE (250 mL) at 0 ° C. The resulting mixture was transferred to a 2.7 cm diameter filtration column equipped with a 20 μm pore size filter. Filtration was carried out under a pressure of 50 mbar. It took 3 minutes and 45 seconds to filter the entire amount (260 mL) of the precipitated mother liquor. The height of the cake after filtration was 3.5 cm, and from this, a filtration coefficient (K) = 848 was obtained. The solid was collected and dried under reduced pressure. Peptide 4.5 g was isolated as a solid.

  FIG. 2 shows a photograph of the isolated peptide (magnification 40 times).

  The aqueous layer and precipitated mother liquor obtained by the extraction process were analyzed by HPLC. The amount of peptide detected was less than 0.5% by weight of the total amount of peptide contained in 25 mL of the reaction mixture obtained in Example 4.

b) Comparative example: Effect of addition of DMF to the precipitation mother liquor Extraction and precipitation were carried out in the same manner as in a) above except that DMF (2.5 mL) was added to the precipitation mixture before the peptide was filtered. The solid precipitate immediately formed a rubbery solid and could not be filtered.

c) Comparative example: direct precipitation in DIPE
While stirring DIPE (250 mL) at 0 ° C., Boc-Ser (Bzl) -Phe-Pro-Ile-Leu-Pro-Pro-Glu (OBzl) -Glu (OBzl) -Tyr (Bzl) -Leu (OBzl) 25 mL of the reaction mixture obtained in Example 4 containing 5 g was added dropwise to form a precipitate. Peptide precipitates were obtained as sticky rubbery solids. After bilayer separation, the supernatant was aspirated with a pump and replaced with a second batch of DIPE (250 mL). The resulting mixture was stirred for 1 hour to break up the aggregation of sticky rubber-like solids. After bilayer separation, the supernatant was replaced again with DIPE (250 mL) for the third batch. The mixture was stirred again for 1 hour and then transferred to a filtration column. However, most of the solids remain in the form of sticky rubbery solids while remaining attached to the precipitation vessel, and cannot be moved. Filtration of the mother liquor took 2 minutes 30 seconds, and the cake height was 1.75 cm. As a result, a filtration coefficient (K) = 636 was obtained. The collected solid was dried under reduced pressure.

  2.45 g of peptide was isolated.

d) Comparative example: direct precipitation in water
While stirring water (250 mL) at 0 ° C, Boc-Ser (Bzl) -Phe-Pro-Ile-Leu-Pro-Pro-Glu (OBzl) -Glu (OBzl) -Tyr (Bzl) -Leu (OBzl) 25 mL of the reaction mixture obtained in Example 4 containing 5 g was added dropwise to form a precipitate. A very thin layer of precipitate was obtained, which was then transferred to a filtration column. The filtration rate was very slow (<3 mL / h), with a large amount of precipitate passing through the filter in the first stage of filtration, and apparent filter clogging after about 65 minutes. Furthermore, a clear precipitation supernatant was not obtained. Therefore, it was impossible to recover the obtained precipitate.

result
Peptide Boc-Ser (Bzl) -Phe-Pro-Ile-Leu-Pro-Pro-Glu (OBzl) -Glu (OBzl) -Tyr (Bzl) -Leu (OBzl) in the presence of DMF Examples b) to d)), rubbery solids were formed. This solid was difficult to handle. In Example a), DMF contained in a trace amount was substantially removed by extraction, and then the peptide Boc-Ser (Bzl) -Phe-Pro-Ile-Leu-Pro-Pro-Glu (OBzl) -Glu ( OBzl) -Tyr (Bzl) -Leu (OBzl) was precipitated. The resulting product could be isolated in good yield and easily filtered.

  By using the extraction method of the present invention, polar aprotic solvents such as DMF can be efficiently separated from peptides. Thus, the undesirable effects of polar aprotic solvents such as inhibiting the peptide precipitation process can be eliminated.

Claims (16)

A method for extracting a peptide from a reaction mixture obtained by a peptide coupling reaction,
The reaction mixture comprises a peptide and a polar aprotic solvent selected from the group consisting of N, N-dimethylformamide, N, N-dimethylacetamide and N-methyl-2-pyrrolidone;
The method is
a) adding to the reaction mixture component a1) which is toluene and component a2) which is water to obtain a two-phase system consisting of an organic layer and an aqueous layer, and b) an organic layer containing a peptide Including a step comprising separating the aqueous layer; and the volume composition ratio of the two-phase system obtained in step a)
A polar aprotic solvent: toluene = 1: 20 to 1: 2, and a polar aprotic solvent: water = 1: 20 to 1: 2. In step a), component a3) which is an organic solvent 1 selected from the group consisting of n-heptane, 2-methyltetrahydrofuran, ethyl acetate, isopropyl acetate, acetonitrile and tetrahydrofuran is further added to the reaction mixture, the organic layer and Obtaining a two-phase system comprising an aqueous layer; and the volume composition ratio of the two-phase system obtained in step a)
Polar aprotic solvent: toluene = 1: 20-1: 2,
The method according to claim 1, wherein polar aprotic solvent: organic solvent 1 = 1: 5 to 30: 1 and polar aprotic solvent: water = 1: 20 to 1: 2. The volume composition ratio of the two-phase system obtained in step a) is
Polar aprotic solvent: toluene = 1: 6 to 1: 3
3. The method according to claim 2, wherein the polar aprotic solvent: organic solvent 1 = 1: 1 to 4: 1, and the polar aprotic solvent: water = 1: 5 to 1: 3.   4. A method according to one or more of claims 1 to 3, characterized in that the polar aprotic solvent is selected from the group consisting of N, N-dimethylformamide and N-methyl-2-pyrrolidone.   The method according to one or more of claims 2 to 4, wherein the organic solvent 1 is selected from the group consisting of acetonitrile and tetrahydrofuran.   6. The component a2) comprises at least one inorganic salt selected from the group consisting of sodium chloride, sodium hydrogen sulfate, potassium hydrogen sulfate, sodium hydrogen carbonate and sodium hydrogen phosphate. The method according to one or more of the above.   The method according to one or more of claims 1 to 6, wherein the pH of said component a2) is 5-8.   The method according to one or more of the preceding claims, characterized in that the filtration of the two-phase system obtained in step a) is performed before step b).   The method according to one or more of claims 1 to 8, characterized in that step a) and step b) are carried out at 20-30C. A method for preparing a peptide in a liquid phase, comprising:
aa) In the presence of a coupling reagent, the peptide coupling reaction is carried out in a polar aprotic solvent selected from the group consisting of N, N-dimethylformamide, N, N-dimethylacetamide and N-methyl-2-pyrrolidone. Performing steps;
bb) extracting the obtained peptide by the method according to one or more of claims 1 to 10; and cc) evaporating at least part of the organic layer obtained in step bb).   11. The method of claim 10, wherein the coupling reagent is selected from the group consisting of O-1H-benzotriazole uronium salts, O-1H-benzotriazole phosphonium salts and carbodiimide coupling reagents.   12. The peptide coupling reaction in step aa) is performed in the presence of a tertiary base selected from the group consisting of N, N-diisopropylethylamine, triethylamine and N-methylmorpholine. The method described in 1. dd) mixing the organic layer obtained in step cc) with an organic solvent 2 selected from the group consisting of acetonitrile, diethyl ether, diisopropyl ether and n-heptane;
13. The method according to one or more of claims 10 to 12, further comprising: ee) precipitating at least a majority of the peptide; and ff) filtering the precipitated peptide.   When the N-terminal protecting group of the peptide is a tert-butyloxycarbonyl protecting group, the tert-butyloxycarbonyl protecting group is removed by treating the organic layer obtained in step cc) with trifluoroacetic acid. The method according to one or more of claims 10 to 12, characterized in that   When the N-terminal protecting group of the peptide is a fluorenyl-9-methoxycarbonyl protecting group, the reaction mixture obtained by the peptide coupling reaction of step aa) is treated with piperidine to protect the fluorenyl-9-methoxycarbonyl protection. 13. A method according to one or more of claims 10 to 12, wherein the group is removed.   The C-terminal carboxylic acid group of the peptide is protected with 2-chlorophenyldiphenylmethyl ester or N-methyl-9H-xanthene-9-amide, according to one or more of claims 10-15 Method.